We propose to develop a method for direct real-time sequencing of single DNA molecules from genomic DNA at the speed and accuracy of the natural DNA polymerases using native nucleotides. We will harness the power of the true nano-machines used in DNA replication, the natural DNA polymerases. Unlike the difficult to engineer man-made nanostructures of nanopore sequencing used to distinguish the 4 base types in close proximity and constant fluctuation, DNA polymerases have precise atomic-resolution 3D structures and can synthesize very long DNA molecules with high fidelity and velocity. The error rate of a DNA polymerase with proof-reading function could be as low as one in a million bases and a processive polymerase such as phi29 DNA polymerase can synthesize up to 100,000 bases in a stretch. From the wealth of structural and kinetics studies, it is well known that the fidelity of DNA synthesis is predicated on the exquisite structural complementarity and the numerous specific interactions between the active site of the polymerase protein and the primer/template/nucleotide complex. The dynamic chemo-mechanical or conformational changes accompanying the specific interactions, induced fit, bond cleavage/formation, and template translocation ensure highly accurate and orderly base pairing and incorporation. Our strategy is to engineer sensors onto the surface (not the active site) of the polymerase by protein engineering to monitor the subtle yet distinct conformational changes accompanying the incorporation of each base type. A small distance change (one to tens of angstroms) can be measured precisely with F"rster resonance energy transfer (FRET) technique. Multiple FRET pairs or networks placed in strategic residues on the polymerase will be used to monitor the conformational changes in real time (10 times faster than the rate of DNA synthesis). The sensors will provide multi-parametric information on the dynamic structures of the polymerase, which very likely will provide a unique signature for each base type incorporated. Chemical modifications such as methylation on the template DNA could also potentially be detected. Such a method could sequence very long DNA molecules and could be sequenced with high fidelity in minutes and a human genome or even epigenome could be sequenced in less than one hour. This will truly enable personalized medicine. PUBLIC HEALTH RELEVANCE: We propose to develop a breakthrough DNA sequencing technology called READS genome technology for direct real-time single molecule sequencing. We aim to develop the new sequencing method and engineer a sequencing platform for ultra-fast and low-cost human genome sequencing so that routine sequencing of individual human genomes can be performed for biomedical applications and personalized medicine.